Influenzavirusisaseriouspublichealththreatcausingsignificantmorbidityandmortality.Seasonalinfections are punctuated by pandemic outbreaks with the potential for widespread infection and disease. This is exemplifiedbytheemergenceofthe2009H1N1pandemicvirusthatrapidlybecamethedominantcirculating strain.Inspiteofthisrisk,thereareonlytwomainclassesofapprovedantivirals,andonlytheneuraminidase inhibitors are efficacious against currently circulating strains. There is a clear need for the discovery of new antiviral therapies and the identification of novel antiviral targets. The influenza virus polymerase is an attractive target for antiviral development. The polymerase assembles with genomic RNA and the viral nucleoprotein(NP)toformlargeribonucleoprotein(RNP)complexes.TheRNPsmediatebothtranscriptionof viralmRNAandreplicationoftheviralgenome.RNPassemblyandpolymeraseactivityareessentialforviral replication, yet how these events are regulated has long remained unclear. We have generated data suggesting that post-translational modifications of NP and the viral polymerase dynamically regulate the formationofRNPs,andtheresultantsynthesisofviralRNAs.Theoverallgoalofthisproposalistounderstand howpost-translationalmodificationsregulateinfluenzavirusRNPassemblyandpolymeraseactivity.Ourfirst objective (Aim 1) is to establish the mechanisms by which phosphorylation regulates NP oligomerization. TheseexperimentsidentifyhostkinasesandphosphatasesthatregulateNPphosphorylationanddemonstrate howthiscontrolsthetransitionofNPfromamonomertothehigher-orderoligomerpresentintheRNP.Wewill alsodefinethetemporalpatternsofNPphosphorylationandhowtheseimpactthetransitionfromtranscription ofviralmRNAstoreplicationoftheviralgenome.Oursecondobjective(Aim2)istodeterminethefunctional consequencesofnovelpost-translationalmodificationswerecentlyidentifiedontheviralpolymeraseandNP. Experiments will determine the host factors mediating this modification and the biological impact during infections.CompletionofthisaimwilldefineacompletelynewregulatorymechanismcontrollingRNPactivity. Theresultsfromthisproposalwillestablishamechanisticunderstandingoftheviralandhostfactorsregulating assembly and function of the RNP and identify exciting new targets that can be exploited for the rational developmentofanti-influenzavirustherapies.